Genetic diversity and multiplicity of infection in Fasciola gigantica isolates of Pakistani livestock

Abstract

Fasciola gigantica liver flukes are responsible for over 3 billion US dollars of production loss annually in farmed livestock and cause widespread zoonotic disease. Nevertheless, the understating of the emergence and spread of the trematode species is poor. The multiplicity of F. gigantica infection and its spread is potentially influenced by multiple factors, including the abundance of suitable intermediate hosts, climatic conditions favoring the completion of the parasite's lifecycle, and translocation of infected animals or free-living parasite stages between regions. Here we describe the development of a 'tremabiome' metabarcoding sequencing method to explore the numbers of F. gigantica genotypes per infection and patterns of parasite spread, based on genetic characteristics of the mitochondrial NADH dehydrogenase 1 (mt-ND-1) locus. We collected F. gigantica from three abattoirs in the Punjab and Balochistan provinces of Pakistan, and our results show a high level of genetic diversity in 20 F. gigantica populations derived from small and large ruminants consigned to slaughter in both provinces. This implies that F. gigantica can reproduce in its definitive hosts through meiosis involving cross-and self-breeding, as described in the closely related species, Fasciola hepatica. The genetic diversity between the 20 populations derived from different locations also illustrates the impact of animal movements on gene flow. Our results demonstrate the predominance of single haplotypes, consistent with a single introduction of F. gigantica infection in 85% of the hosts from which the parasite populations were derived. This is consistent with clonal reproduction in the intermediate snail hosts.

Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan

“…There were both unique and common haplotypes in each D. dendriticum population from the Chitral valley, some of which were also present in populations form China, Iran and Japan. There are insufficient data on which to base firm conclusions; hence further studies based on larger population sizes, and using for example next generation methods as described for Calicophoron daubneyi rumen flukes and F. gigantica liver flukes [16,17] are needed to describe gene flow and the role of animal movement in the spread of D. dendriticum.…”

“…Molecular methods amplifying fragments of nuclear ribosomal genes and their internal transcribed spacers, or mitochondrial loci DNA [8,9,10,11,12,13] have been developed for Dicrocoeliid parasites; but as with all molecular diagnostic tools, these depend on accurate morphological speciation of the reference materials. These methods are adaptable to demonstrate genetic variability and phylogeny, and have been applied to epidemiological studies of various trematode parasite species affecting ruminant livestock [14,15,16,17]. However, in the study of Dicrocoeliid genera, the value of phylogenetic studies to detect intraspecific variation is limited by the availability of comparable sequence data.…”

Towards the comprehension of fasciolosis (re-)emergence: an integrative overview

“…mitochondrial or nuclear) in Spain (Thang et al ., 2020), Austria (Husch et al ., 2020), Armenia (Aghayan et al ., 2019), Iran (Bozorgomid et al ., 2020) and Australia (Elliott et al ., 2014). In the case of F. gigantica , high genetic diversity is also reported from Pakistan (Rehman et al ., 2020), Cambodia (Loeurng et al ., 2019) and Nigeria (Ichikawa-Seki et al ., 2017 b ) using mitochondrial markers. However, other countries have shown lower haplotype diversity generally associated with recent introductions from particular regions, such as in Indonesia (Hayashi et al ., 2016).…”

High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan, but only Fasciola gigantica in the Punjab province of Pakistan

“…in Pakistan. Some report F. hepatica to be most prevalent in ruminants (Ahmad et al, 2017;Akhtar et al, 2012;Ijaz et al, 2009;Shahzad et al, 2012), while others indicate a greater prevalence of F. gigantica (Bhutto et al, 2012;Khan et al, 2009;Khan et al, 2010;Maqbool et al, 2002;Rehman et al, 2020). These studies are all based on morphological Fasciola spp.…”

“…High throughput next generation sequencing methods, using platforms such as the Illumina Mi-Seq now provide a relatively low-cost and reliable opportunitiy to study the epidemiology of Fasciola spp. (Rehman et al, 2020). Ribosomal and mitochondrial DNA markers such as the internal transcribed spacer-2 (ITS-2) and nicotinamide adenine dinucleotide dehydrogenase-1 (ND-1) sequence reads allow differentiation between Fasciola species and hybrids, and can be used to describe genetic diversity and multiplicity of infection (Ai et al, 2010;Amor et al, 2011;Chaudhry et al, 2016;Rehman et al, 2020;Rokni et al, 2010).…”